In recent years, functional nucleic acids controlling the expression of particular genes in vivo have received attention as novel pharmaceutical drugs or diagnostic drugs comparable to compounds and antibodies. Various studies and developments toward medical applications thereof are underway around the world.
The known functional nucleic acids include, for example: small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), and micro RNAs (miRNAs), which post-transcriptionally suppress the expression of target genes by gene silencing mediated by RNA interference (RNAi); nucleic acid aptamers, which suppress the functions of target substances such as transcription factors by specifically binding thereto; antisense nucleic acids, which suppress the translation of target mRNAs by binding thereto; decoy DNAs containing regulatory regions such as transcription factor-binding domains as decoy sequences, wherein the decoy DNAs capture target substances, thereby suppressing gene expression caused by the transcription factors; and U1 adaptors, which specifically inhibit polyadenylation in the mRNA precursors of target genes to destabilize the mRNA molecules and then direct the degradation thereof. All of them are expected as the next-generation pharmaceutical drugs or diagnostic drugs. Among them, RNAi by siRNAs or shRNAs is in the limelight as powerful gene expression control tools capable of suppressing the desired gene expression, because of their target specificity, wide applications, and reliable functions or effects.
Allele-specific gene silencing (or allele-specific RNAi: ASP-RNAi), which is an application of RNAi, can specifically suppress the expression of a desired allele. This technique can specifically suppress the expression of a target dominant mutant gene causative of a disease without influencing the expression of the wild-type gene and as such, is considered exceedingly useful in the therapy of the disease. For example, fibrodysplasia ossificans progressiva (FOP) known as an intractable autosomal dominantly inherited disease is caused by a point mutation that substitutes guanine (G) at position 617 by adenine (A) or a point mutation that substitutes G at position 1067 by A on its causative activin-like kinase 2 (ALK2) gene. Since a mutant gene having any of these point mutations is dominant, even a heterozygote having the wild-type ALK2 gene develops FOP (Non Patent Literatures 1 to 3). Unfortunately, an effective method for preventing the onset or progression of FOP has not yet been found. In this regard, if ASP-RNAi can suppress only the expression of a dominant mutant gene and permit the expression of the wild-type gene, the onset of autosomal dominantly inherited diseases including FOP can be prevented. In addition, the progression of these diseases can be prevented for patients who have already developed the diseases. Thus, ASP-RNAi molecules, among the RNAi molecules, are particularly highly useful as pharmaceutical drugs or diagnostic drugs.
Since such a base-substitution mutant gene having a point mutation differs from the wild-type gene in their nucleotide sequences only by one or several bases, conventional RNAi molecules based on general design methods suppress the expression of the wild-type gene due to their low specificity for the mutant gene. Even if a mutant gene has a clear difference from the wild-type gene in their nucleotide sequences, as in a dominant mutant gene that results in a transcript containing a point of discontinuity, the RNAi molecules designed by the conventional methods do not always have high specificity for the mutant gene and may often suppress the expression of the wild-type gene. Thus, the development of siRNAs or shRNAs having exceedingly high specificity for mutant genes is essential for achieving such ASP-RNAi. Nevertheless, the design of siRNAs or the like is inevitably limited by design region, because a mutation site (e.g., a substitution, deletion, or insertion site) and its neighboring nucleotide sequences must be used as the target region. Hence, the design may disadvantageously fail to constantly produce highly specific and effective siRNAs or the like, even by the application of effective methods for selecting target sequences of siRNAs known in the art.